Vegetable protein fraction with phospholipase d activity

ABSTRACT

What is claimed is a novel plant phospholipase D activity-containing protein fraction which is derived from representatives of the Papaveraceae family, and, in particular, from  Papaver somniferum , and which is composed of two protein subfractions A and B whose molecular masses are, in particular, 116.4 kDa and, respectively, 114.1 kDa, which have isoelectric points, pI, of 8.7 and, respectively, 6.7 and which have hydrolytic activity optima at pH 8.0 and, respectively, 5.5. The two subfractions are activated by Zn 2+  ions, in particular, and constitute two isoenzymes. This protein fraction is used, in particular, for hydrolyzing and/or transphosphatidylating phospholipids and their lyso forms.

The present invention relates to a plant protein fraction whichpossesses phospholipase D activity.

Phospholipase D (PLD), which is a phosphatidylcholine choline hydrolase(EC 3.1.4.4), is an important enzyme of phospholipid metabolism and iswidespread in nature.

Phospholipase D is assigned to an enzyme class whose representatives areable, in heterogeneous systems, to transform water-insoluble substratessince they are able to catalyze reactions at the interface between alipid and water. The fact that phospholipase D exhibits this amphiphilicbehavior has resulted in this enzyme appearing to be of particularinterest for science, such that a large number of differentphospholipases D (species) could be isolated from a very wide variety ofsources during the past decades. In particular, PLD is thought to beinvolved in cell-regulatory activities in connection with theintercellular signal exchange. PLD enzymes are also able, by way oftheir hydrolysis activity, to transfer phosphatidyl residues toalcohols. In a general manner, therefore, PLD variants are employed forthe biocatalytic exchange of phospholipid head groups.

Thus, for example, enzyme fractions are known from sugarbeet, spinach orcabbage leaf plastids and from carrot chromoplasts. Correspondingfractions possessing phospholipase D activity have also been isolatedfrom mitochondria and microsomes of immature peanut seeds as well asfrom castor oil seeds, Arabidopsis species and tomatoe.

Success has also been achieved in extracting a corresponding enzyme fromdefatted cottonseed meal.

In addition to plant sources, microorganisms also serve as a source,whereas, in particular, corynebacteria (Corynebacterium ovis),Escherichia coli, baker's yeast cells and streptomycetes (Streptomyceshachijoensis) are to be mentioned.

However, it has also been possible to isolate phospholipase frommammalian cells, for example from human eosinophils and rat brainmicrosomes.

The known phospholipase D enzymes present a heterogeneous picture inregard to their molecular weights:

Thus, the soluble enzyme isolated from cotton seed has a molecularweight of 71 000 3000 Da; phospholipase D from peanut seeds has amolecular weight of 200 000 10 000 Da and PLD from human eosinophils hasa molecular weight of approx. 60 000 Da.

Corresponding bacterial enzymes, as can be isolated, for example, fromCorynebacterium ovis, possess a molecular weight of approximately 90 000Da.

With regard to the isoelectric point, phospholipase D from peanut seedsis known to have pI values of 4.65 while, on the other hand, the pI of acrude extract from human eosinophils is between 4.8 and 5.0 and, as aresult of additional purification, can reach a value of between 5.8 and6.2.

R. Lambrecht et al. (“A facile purification procedure of phospholipase Dfrom cabbage and its characterization”; (1992) Biol. Chem. Hoppe SeylerVol. 373 (2) 81-88) describe purification of PLD from white cabbage intwo steps. This method comprises an ammonium sulfate precipitation and asubsequent Ca⁺-mediated affinity chromatography.

The publication by I. Schaffner et al. (“Genomic structure, cloning andexpression of two phospholipase D isoenzymes from white cabbage”, Eur.J. Lipid Sci. Technol. 104, 79-87 (2002); corresponding to dissertation(2001)) describes how recombinant phospholipase D-active isoenzymes canbe obtained from white cabbage using cloning methods and how theseisoenzymes can be characterized in regard to their specific hydrolysisactivity in dependence on the pH and on the Ca²⁺ concentration as wellas in regard to their transphosphatidylating properties.

The review article by Michael Heller in Advanced Lipid Research, 1978,Volume 16, pages 267 to 326, provides a general overview of the state ofknowledge with regard to phospholipase D.

In “Identification of two isoenzymes of phospholipase D from opiumpoppy” (Direct submission (2001) NCBI GenBank, accession Nos.AAL48261-AAL48264 and multiple sequence comparison), A. Lerchner et al.describe two truncated phospholipase D1 polypeptides, as well as twoother truncated phospholipase D2 polypeptides, from Papaver somniferum.As can be seen from the multiple sequence comparison, the part aminoacid sequences of proteins D1 and D2 exhibit a sequence identity of 98%to each other. In addition, the part sequences which are describedpossess a high degree of homology (70-84%) with the well-characterizedphospholipase D varieties of the type. However, since the sequencedetermination is not complete at the 5′ end, it is not possible toassign the phospholipase D1 and D2 polypeptides to a defined enzyme.

The database entry by A. Lerchner et al. “Identification of twoisoenzymes of phospholipase D from opium poppy” (Direct submission(2001) NCBI GenBank, accession Nos. AF451979-AF451982) describes nucleicacid sequences for the two polypeptides PLD1 and PLD2 which have justbeen mentioned.

Poppy seeds are known to be able to form secondary metabolites on anunusually large scale. Thus, alkaloids such as thebaine can, forexample, be detected in poppy seeds after only a few days of swelling,thereby making the seeds of interest with regard to opium isolation, inparticular.

Since phospholipase D (PLD) plays an evermore important role in theindustrially employed catalytic hydrolysis of glycerophospholipids suchas phosphatidylcholine (PC) to phosphatidic acid (PA), and also intransphosphatidylating processes in regard to headgroup exchange inphospholipids, the object of the present invention was that of isolatingnovel plant protein fractions which originate from representatives ofthe Papaveraceae family and which possess phospholipase D activity.

This object was achieved by means of a corresponding protein fractionwhich is characterized in that

-   -   a) it consists of two protein subfractions A and B, and    -   b) it can be activated by Zn²⁺ ions, and also    -   c) the subfractions A and/or B possess carbohydrate moieties,        and with the protein subfraction A only possessing hydrolysis        activity.

In accordance with the definition, the term “protein fraction” which isemployed below encompasses all actual protein fractions and proteins aswell as their possible variants, and also all enzymes and enzymevariants, all of which possess corresponding PLD activity.

Surprisingly, it was possible to establish that this plant proteinfraction contains two isoenzyme units which both exhibit a relativelynarrow molecular mass spectrum and whose activity optima lie in thestrongly acidic region, on the one hand, but in the slightly basicregion on the other hand. In addition, it was not to be expected, on thebasis of the previously known PLDs, that isoenzymes from poppypossessing PLD activity can be activated with zinc, something which isadvantageous particularly in regard to their use for preparingphospholipids, which are known to form mostly insoluble complexes withCa ions. In addition, it was not possible to assume, on the basis ofpreviously available findings with phospholipases D from plant sources,that protein fractions with corresponding activities will be found inrepresentatives of the Papaveraceae family.

The present invention claims, in particular, a protein fraction which isderived from Papaver somniferum (opium poppy) and very particularlypreferably from developing seedlings and/or endosperms. It is naturallyalso possible to conceive of a variant in which the protein fractionsare derived indirectly from Papaver in that the protein fractions areobtained namely by means of recombinant methods, in particular usingrecombinant microorganisms which contain the genes for the correspondingprotein fraction.

As already explained, one essential aspect according to the invention isdirected towards the fact that the claimed plant protein fractioncontains two isoenzymes. In this regard, the present invention prefers aprotein fraction whose subfraction A possesses a molecular mass between116 and 118 kDa, an isoelectric point pI between 8.5 and 8.9 and ahydrolytic activity optimum at pH values between 7.8 and 8.2, and thesubfraction B subfraction B possesses a molecular mass between 112 and115 kDa, an isoelectric point pI between 6.5 and 6.9 and a hydrolyticactivity optimum at pH values between 5.0 and 6.0.

A defined value for the isoelectric point can be obtained by subjectingisolated fractions to further purification.

For this reason, the protein fraction of the present invention is also,in particular, characterized by the fact that the subfraction A has anisoelectric point pI of 8.7 and a molecular mass of 116.4 kDa as well asa hydrolytic activity optimum at pH 8.0. The corresponding preferredvalues for subfraction B are 114.1 kDa with regard to the molecular massand 6.7 with regard to the isoelectric point pI, with the hydrolyticactivity optimum being at pH 5.5. These features are also encompassed bythe present invention.

As already mentioned, protein fractions possessing phospholipase Dactivity are usually calcium ion-dependent. However, this pronounceddependence has not proved to be true in the case of the claimed plantprotein fraction from Papaveraceae, which is imperatively Zn²⁺ion-activatable. However, the activity optimum of this protein fractioncan also be reached in the presence of calcium ion concentrations, whichare then usually between 40 M and 100 mM, with corresponding enzymeactivities appearing at concentrations of between 2 and 20 mM andbetween 5 and 15 mM.

With regard to subfraction B, the present invention claims a proteinvariant whose activatability optimum occurs in the presence of Zn²⁺ ionconcentrations which are between 1.0 and 10 mM and, particularlypreferably, at 5 mM.

As what is essential for the invention, inter alia, the presentinvention provides for subfractions A and/or B to possess carbohydratemoieties such that they are consequently present in glycosylated form asN-linked glycoproteins, and for subfractions A and B to be isoenzymes.

In conformity with the surprisingly different activity properties ofsubfractions A (only hydrolysis) and B (pronouncedtransphosphatidylation), the present invention also encompasses avariant of the protein fraction in which the transphosphatidylationactivity is, all in all, more strongly expressed than its hydrolysisactivity, something which can be explained, in particular, by theindividual activities of the subfractions and is also of importance withregard to the previously known PLD variants, as compared with which thenovel protein fractions exhibit transphosphatidylation activities whichare up to 100 times more pronounced, based on the correspondinghydrolysis activities.

In addition to the protein fraction itself, the present invention alsoclaims the use of this protein fraction for hydrolyzing and/ortransphosphatidylating phospholipids and/or their lyso forms, with, inparticular, the synthesis of phosphatidylcholine,phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol,phosphatidic acid and phosphatidylserine, and their lyso forms and anyarbitrary mixtures, being claimed. In this connection, it is also worthmentioning the fact that the protein fraction according to the inventionis able to hydrolyze phosphatidylinositol and/or to carry out aheadgroup exchange on PI, something which the previously known PLDs arelikewise not known to do. While, in this connection, the conduct of thereaction as a whole is not to be regarded as being critical, organicand/or aqueous phases have proved to be very suitable as reaction mediawhile phosphatidylcholine and phosphatidylethanolamine have proved to bevery suitable as the phospholipid source.

In summary, it can be stated that, by means of this novel phospholipaseD activity-possessing plant protein fraction obtained fromrepresentatives of the Papaveraceae family, success has been achieved inisolating a PLD which, taken overall, possesses a very pronouncedtransphosphatidylation activity and whose two isoenzymic subfractionsexhibit hydrolytic activities, in particular towardsphosphatidylcholine; while both subfractions can be activated by calciumions, as is a known property of PLDs, the novel protein fraction can, incontrast to known phospholipase D variants from other plants, also, andin particular, be activated by Zn²⁺ ions.

The novel protein fraction consequently differs fundamentally from thepreviously known plant PLDs and its properties differ markedly fromthose of the PLDs whose gene sequences have already been determined.

The following examples clarify the characteristic features of theclaimed plant protein fraction possessing phospholipase D activity.

EXAMPLES

Working-Up of Plant Material (Enzyme Isolation)

Poppy seeds (Papaver somniferum), which were present on a 10 mm thickpolyurethane foam layer in Petri dishes which were covered with a nylonfabric, were germinated in distilled water. The germination process wascarried out in the dark, at 25° C. and at from 70 to 80% relativehumidity. On the second day after the swelling, the endosperm wasremoved from the seedlings.

These endosperms, which were obtained from a total of 10 g of freshpoppy seeds, were triturated with a small quantity of cold acetone in amortar and then homogenized with 300 ml of cold acetone which contained300 g of solid CO₂. The resulting precipitate was then washed with coldacetone until the filtrate was colorless and transparent. In powderform, the vacuum-dried residue was stable for several months at 4° C.

2 g of this acetone powder were homogenized in 50 ml of a mixtureconsisting of 0.1 molar sodium acetate buffer/10 mM CaCl₂/6 mM cysteinehydrochloride (pH 5.5) and centrifuged at 12 000 g and 4° C. for 10minutes. The resulting extracts were treated with (NH₄)₂SO₄ (60%saturation) and centrifuged at 26 000 g and 4° C. for 45 minutes. Theprecipitate was then taken up in a preferably minor quantity of amixture, consisting of 0.01 sodium acetate buffer/10 mM CaCl₂/6 mMcysteine hydrochloride (pH 5.5). After dialysis against a mixturecomposed of 0.01 molar sodium acetate buffer/50 mM CaCl₂ (pH 5.5), theenzyme solution was loaded onto an octyl-Sepharose CL-4B column. Theproteins were eluted at a flow rate of 9 ml/h in three steps using thefollowing solutions: 0.01M sodium acetate buffer/50 mM CaCl₂; 0.005 Msodium acetate buffer/30 mM CaCl₂ (pH 5.5). 0.005 M sodium acetatebuffer/0.1 mM ethylenediaminetetraacetic acid (EDTA), pH 5.5. The enzymeactivity was determined at pH values of 5.5 and 8.0 usingphosphatidyl-p-nitrophenol (PpNP), and the combined active fractionswere concentrated using a 100 kDa membrane.

Protein Determination

The protein content was determined in accordance with the standardmethod of M. M. Bradford (Anal. Biochem. 72, 1976, 248-254) using bovineserum albumin as standard.

Hydrolytic Activity in Aqueous Systems

The hydrolytic activity of the resulting plant protein fractionpossessing phospholipase D activity was determined in an aqueous systemby determining the p-nitrophenol which was released from PpNP (Method inaccordance with P. D'Arrigo, V. Piergianni, D. Scarcelli, S. Servi, “ASpectrophotometric Essay for Phospholipase D”, Anal. Chim. Acta, 304,1995, 249-254).

In order to characterize the respective activities of the PLDsubfractions A and B, the reactions were carried out at different pHvalues in the presence of 10 mM CaCl₂, in the presence of differentconcentrations of CaCl₂ at pH values of 5.5 and, respectively, 8.0, andalso in the presence of different concentrations of ZnCl₂ at a pH of5.5.

Transphosphatidylation and Hydrolytic Activity in a Two-Phase System

In conformity with the method according to N. Dittrich and R.Ulbrich-Hofmann (“Transphosphatidylation by immobilised Phospholipase Din aqueous media”, Biotechnol. Appl. Biochem. 34, 2001, 189-194), thetransphosphatidylation and hydrolysis activities were determined in atwo-phase system. The corresponding reaction media were composed of aphosphatidylcholine-containing diethyl ether, glycerol (for determiningthe transphosphatidylation activity) or water (for determining thehydrolysis activity) as well as a mixture composed of Tris-HCl/CaCl₂ ora mixture composed of sodium acetate buffer/CaCl₂ and a purified enzyme(PLD-A and PLD-B). The respective reactions were carried out at 30° C.,and at a shaking frequency of 250/min, in reaction vessels which weresealed with Teflon-silicone septa. During the reaction, aliquots of theorganic phase(s) were analyzed by means of HPTLC. The phospholipidcontents were determined densitometrically at 550 nm in comparison withstandard mixtures composed of phosphatidylcholine, phosphatidic acid andphosphatidylglycerol, with the enzyme activity being calculated from theincrease in phosphatidyl-glycerol or phosphatidic acid.

Determining Enzyme Features

The molecular mass of the purified proteins was determined by means ofelectrophoresis in the presence of SDS using a Bio-Rad Mini Protein IIgel electrophoresis cell and polyacrylamide gels.

The isoelectric points of the PLD subfractions A and B were determinedusing a PhastGel IEF 3-9 on a FastSystem separation and control unit(Pharmacia. LKB Biotechnology), with pI IEF markers (liquid mix 3-10)being employed for the calibration. The proteins were stained withCoomassie Brilliant Blue G-250.

For the glycoprotein determination, an SDS-PAGE gel which containedPLD-A, PLD-B and peroxidase (as standard for a glycosylated protein) andalso aldolase (as standard for a nonglycosylated protein) was broughtinto contact, at 300 V and 5 mA/cm², with a nitrocellulose membrane fora period of 180 minutes. After the proteins had transferred, the totalcarbohydrate content was determined using an ECL glycoprotein detectionmodule.

The N-terminal amino acids were sequenced using a 492 cLC proteinsequencer (PE Applied Biosystems).

Results:

The two enzyme forms which were obtained by means of hydrophobicinteraction chromatography for purifiying PLD, in accordance with R.Lambrecht and R. Ulbrich-Hofmann (“A facile purification procedure ofphospholipase D from cabbage and its characterization”, Biol. Chem.Hoppe-Seyler 373, 1992, 81-88), were active at pH 8.0 (PLD-A) and,respectively, pH 5.5 (PLD-B). The same purification results could beachieved by replacing CaCl₂ with ZnCl₂ in buffer solutions. Both enzymesubfractions were homogeneous in an SDS-PAGE gel. Table 1 shows thepurification data, with the purification factors for the two isoenzymesbeing 84.7 (PLD-A) and, respectively, 94.1 (PLD-B).

Protein Determination of the Two Subfractions

Using the SDS-PAGE method, the molecular masses of PLD subfraction A andPLD subfraction B were determined to be 116.4 and, respectively, 114.1kDa. Their isoelectric points were 8.7 (PLD-A) and 6.7 (PLD-B). It wasdemonstrated that both PLD-A and PLD-B were present in glycosylated formsince a positive deglycosylation reaction using N-glycosidase F showedthe presence of an N-bound carbohydrate in the case of bothsubfractions. Since the N-terminal sequencing method failed in the caseof both subfractions, it is to be assumed that an N-terminalmodification is present in both cases.

pH Activity Profiles

Significant differences were found in the hydrolytic activities of PLDsubfractions A and B toward PpNP and as a function of the pH.Subfraction A possesses a sharp pH optimum at pH 8.0 while subfraction Bhas no particularly marked activity at this pH. By contrast, the pHoptimum of subfraction B is at pH 5.5, at which subfraction A scarcelyexhibits any activity. Under conditions which are in each case optimal,subtraction B exhibits an activity which is 38% higher than that ofsubfraction A.

Influence of Metal Ions

In regard to the fact that PLD variants are known to be activated byCa²⁺ ions, the novel subfractions A and B from opium poppy were alsofound to exhibit calcium ion dependency, with an activity maximum beingobtained at a CaCl₂ concentration of 10 mM. It was only possible toobtain very slight activation of the two subunits with Mg²⁺ ions,whereas Zn²⁺ ions activated the PLD subtractions A and, in particular, Bmore strongly than did calcium ions. Subfraction B was activated fourtimes more strongly by an optimal Zn²⁺ ion concentration (5 mM) than itwas by the optimal Ca²⁺ ion concentrations.

Transphosphatidylating Activities and Hydrolytic Activities in a 2-PhaseSystem

The transphosphatidylating potentials of subfractions A and B weredetermined, by means of HPTLC and densitometric quantification of thereaction products, in a biphasic system which was composed of a sodiumacetate buffer (pH 5.5) or Tris-HCl buffer (pH 8.0) and 40 mM CaCl₂,diethyl ether which contained phbsphatidylcholine as substrate andglycerol as acceptor alcohol. At pH 5.5, PLD subtraction B possessedhigh transphosphatidylating potential since more than 80% of thephosphatidylcholine had been converted into the transphosphatidylationproduct phosphatidylglycerol after 240 minutes, whereas it was notpossible to find any phosphatidic acid at all under these reactionconditions. PLD-B did not exhibit any transphosphatidylation orhydrolysis at a pH of 8.0. PLD subtraction A did not exhibit anytransphosphatidylating activity either at pH 5.5 or pH 8.0; as expected,however, it exhibited pronounced hydrolysis activity at pH 8.0 TABLE 1Purification of protein fractions with PLD activity from opium poppyseeds. The hydrolytic PLD activity of the protein fraction towards PpNPwas determined at pH 8.8 and 5.0. Specific activity Activity [μmol min⁻¹Purification Protein [μmol min⁻¹] mg⁻¹] step [mg] pH 8.0 pH 5.5 pH 8.0pH 5.5 Crude extract* 43.25 3.74 4.65 0.09 0.11 (NH₄)₂SO₄ 9.56 1.99 2.380.21 0.25 precipitate** Octyl-Sepharose CL-4B Subfraction A (PLD-A) 0.141.05 — 7.32 — Subfraction B (PLD-B) 0.18 — 1.85 — 10.13*after homogenizing the acetone powder and centrifuging at 12 000 g.**after centrifuging the precipitate at 26 000 g and then dialyzing.

1. A plant protein fraction which is derived from representatives of thePapaveraceae family and which possesses phospholipase D activity,comprising a) two protein subfractions A and B; b) it can be activatedby Zn²⁺ ions, and also c) at least one of subfractions A or B possesscarbohydrate wherein only protein subfraction A possesses hydrolysisactivity.
 2. The protein fraction as claimed in claim 1, derived fromPapaver somniferum and very particularly preferably from developingseedlings or endosperms.
 3. The protein fraction as claimed in claim 1wherein the subfraction A possesses a molecular mass of between 116 and118 kDa, an isoelectric point, pI, of between 8.5 and 8.9 and ahydrolytic activity optimum at pH values of between 7.8 and 8.2, and thesubfraction B possesses a molecular mass of between 112 and 115 kDa, anisoelectric point, pI, of between 6.5 and 6.9 and a hydrolytic activityoptimum at pH values of between 5.0 and 6.0.
 4. The protein fraction asclaimed in claim 1 wherein the subfraction A has a molecular mass of116.4 kDa, an isoelectric point, pI, of 8.7 and a hydrolytic activityoptimum at pH 8.0.
 5. The protein fraction as claimed in claim 1,wherein the subfraction B has a molecular mass of 114.1 kDa, anisoelectric point, pI, of 6.7 and a hydrolytic activity optimum at pH5.5.
 6. The protein fraction as claimed in claim 1, wherein thesubfraction B possesses an activatability optimum at Zn²⁺ ionconcentrations of between 1.0 and 10 mM and, particularly preferably, at5 mM.
 7. The protein fraction as claimed in claim 1 wherein thesubfractions A and B are isoenzymes.
 8. The protein fraction as claimedin claim 1 wherein its transphosphatidylating activity of the proteinfraction is more strongly pronounced than its hydrolysis activity.
 9. Amethod comprising hydrolyzing or transphosphatidylating phospholipids ortheir lyso forms with the protein fraction of claim
 1. 10. The method asclaimed in claim 9 for synthesizing phosphatidylcholine,phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol,phosphatidic acid and phosphatidylserine and their lyso forms.
 11. Themethod as claimed in claim 9 phosphatidylinositol is hydrolized or aheadgroup exchange is performed on phosphatidylinositol.